ITM2A as a Tumor Suppressor and Its Correlation With PD-L1 in Breast Cancer

Abstract

Background: High expression of integral membrane protein 2A (ITM2A) was reported to be associated with favorable prognosis in several solid tumors including breast cancer. This study aimed to investigate the role of ITM2A in breast cancer, especially in respect to tumor microenvironment.

Methods: ITM2A expression was evaluated based on qRT-PCR results on breast cancer specimens, as well as TCGA and GEO datasets. The influence of ITM2A expression on breast cancer cell proliferation and tumor growth were evaluated by CCK-8 assay, clonogenic assay, and murine xenograft models. Transwell assay was performed to observe the changes of invasion and migration capacity in breast cancer cells. To determine the biological functions of ITM2A, differentially expressed genes (DEGs) were screened based on RNA-sequencing data of MCF-7 cells overexpressed ITM2A. Then, functional annotation on DEGs was given by Gene Ontology and KEGG analysis. The stimulation on programmed cell death ligand 1 (PD-L1) expression when ITM2A overexpressed was determined by flow cytometry. Meanwhile, the correlation on expression levels between PD-L1 and ITM2A was tested via qRT-PCR on 24 breast cancer tissues, as well as public database.

Results: We demonstrated that ITM2A was frequently downregulated in breast cancer. Patients with high expression levels of ITM2A had longer overall survival and relapse free survival. Overexpression of ITM2A inhibited proliferation and impaired cells capacity of invasion and migration in vitro and in vivo. The DEGs in breast cancer cells overexpressed ITM2A were found to be associated with immunity responses. Moreover, ITM2A was found to facilitate breast cancer cells to express PD-L1. The correlation between PD-L1 and ITM2A was verified with both qRT-PCR assay and public database. Additionally, it was found that breast cancer had higher ITM2A expression frequently had more tumor-infiltrating lymphocytes (TILs).

Conclusion: In summary, we found that high expression of ITM2A reduced the aggressivity of breast cancer cells and had a favorable effect on outcomes of patients with breast cancer. Moreover, ITM2A induced PD-L1 expression in breast cancer cells was accompanied with higher TILs numbers in tumor microenvironment.

Keywords: ITM2A; PD-L1; breast cancer; immune infiltration; prognosis.

Figure 1

ITM2A was down-regulated in breast cancer and positively associated with favorable outcomes. (A) Comparison of ITM2A expression in breast cancer tissues with that of paired adjacent normal tissues. (B) Boxplot showing expression level of ITM2A in cancer tissues and normal tissues in the breast profile GSE29431 and GSE61304. (C) Comparison of ITM2A expression in cancer tissues with that of paired adjacent normal tissues in the breast TCGA dataset. (D-F) Kaplan-Meier survival curves depicting the OS (D), RFS (E), and DMFS (F) of patients with breast cancer stratified by ITM2A mRNA levels. ***p < 0.001. OS, overall survival; RFS, relapse free survival; DMFS, distant metastasis free survival.

Figure 2

Overexpression of ITM2A inhibited migration and promoted apoptosis of breast cancer cells. MCF-7 and MAD-MB-231 cells were transfected with the indicated plasmid. qRT-PCR (A) and immunoblotting (B) were used to test ITM2A expression in mRNA and protein levels 48 hours after transfection. (C, E) Migration and invasion assay in MDA-MB-231 cells. (D, F) Apoptosis rate in MCF-7 and MDA-MB-231 cells 48 hours after transfection. *p < 0.05, **p < 0.01.

Figure 3

ITM2A overexpression decreased proliferation of breast cancer in vitro and vivo. (A, B) Clone formation was stained with crystal violet 14 days after seeded. (C, D) Growth curves of MCF-7 (left) and MDA-MB-231 cells (right) transfected with the indicated plasmid were measured by CCK-8 assay. (E-G) ITM2A expression inhibited the proliferation of breast cancer cells in vivo. (E) Photographs of dissected tumors from sacrificed mice. (F) Growth curves of indicated tumors in BALB/c null mice. (G) Representative images of enhanced MRI in transverse section. “L” represents the major tumor axis and “W” represents the minor tumor axis. *p < 0.05, **p < 0.01.

Figure 4

Overexpression of ITM2A induced immunity related response. (A) DEGs were derived based on RNA-seq data on MCF-7 cells that overexpressed ITM2A. Then the DEGs were mapped to GO terms and the top 10 ranked GO terms are showed. (B) The top-10 ranked KEGG pathways in which DEGs enriched. DEGs, differentially expressed genes; RNA-seq, RNA-sequencing; GO, gene ontology.

Figure 5

ITM2A increased PD-L1 expression in breast cancer cells. (A) Correlations between PD-L1 and ITM2A expression in lumina breast cancer (left) and basal breast cancer (right) based on TIMER database. (B) Correlation between PD-L1 and ITM2A expression in collected 24 breast cancer specimens. (C, D) MCF-7 and MDA-MB-231 cells were transfected with indicated plasmid. PD-L1 expression in these cells 48 hours after transfection was tested by flow cytometric analysis. ***p < 0.001.

Figure 6

ITM2A expression was positively correlated with TILs quantity. Correlations between ITM2A expression and six TILs types in breast cancer were evaluated based on TIMER database. Analysis in all subtypes of BC (A), basal (B), HER-2 (C), and luminal subtypes (D).